Acanthamoeba castellanii activates murine macrophages in a MyD88 dependent, TRIF and PAR2 independent manner.

Acanthamoeba castellanii is an opportunistic, facultative parasitic protozoan, known to be the agent of a serious, painful, potentially blinding keratitis and fatal encephalitis in humans. Acanthamoeba keratitis is most prevalent in contact lens wearers where it affects 1 in 30,000. Acanthamoeba are ubiquitous in nature, and consequently, although individuals are regularly exposed to these amoebae, very few cases of clinical infection occur, suggesting that the innate immune response is normally effective. Macrophages and neutrophils are predominantly present at the site of infection, implicating them in the resolution of the disease. Herein, macrophage interactions with trophozoites of Acanthamoeba castellanii are investigated in vitro using bone marrow derived macrophages. The role of TLRs and PARs in the recognition and response to Acanthamoeba are also examined using syngeneic mice deficient in MyD88, TRIF or PAR 2 genes. Results demonstrated that Acanthamoeba castellanii stimulated TNF- a , IL-6 and IL-12 production by murine macrophages in a MyD88-dependent, but TRIF-independent manner. Acanthamoeba secreted proteases were able to stimulate the production of pro-inflammatory cytokines by a PAR 2 independent mechanism. In addition, arginase activity was significantly increased in murine macrophages after stimulation with trophozoites, whereas NO production was below detection limits. Taken together, these results suggest that TLR-associated signaling pathways play an important role in initiating the innate immune response to Acanthamoeba castellanii with the ensuing production of pro-inflammatory cytokines and arginase activity.